Infections caused by vancomycin resistant Enterococcus (VRE) are associated with limited treatment options and increased mortality. Daptomycin, a novel lipopeptide antibiotic, has activity against VRE. The daptomycin dose for VRE to optimize patient outcomes and prevent the emergence of resistance, however, is currently unknown. The long-term goal is to optimize outcomes and preserve daptomycin therapy for VRE infections through utilization of the ideal dose exposure and determination of phenotypic and genotypic changes associated with daptomycin resistance in enterococci. The overall objective of this study is to define the dose exposure breakpoint (pharmacokinetic/pharmacodynamic [PK/PD] breakpoint) for daptomycin and the correlating dose and determine if known phenotypic and genotypic changes associated with daptomycin resistance in S. aureus are also found in enterococci. The central hypothesis is that higher daptomycin doses will provide greater bactericidal activity and prevent the emergence of resistance. Additionally, phenotypic and genetic changes found in daptomycin resistant enterococci maybe different than those found in S. aureus. The rationale behind the proposed research is that data on the daptomycin dose relationship with enterococci and insights into the mechanisms of resistance will lead to clinical dose optimization, improved patient outcomes, reduced emergence of resistance, and preservation of daptomycin as a viable antibiotic for clinical use. The central hypothesis will be tested by pursuing two Specific Aims: 1) Determine the dose exposure breakpoints for daptomycin resistance using molecularly defined and clinical strains of VRE to determine the optimal dose; and 2) Identify phenotypic and genetic changes associated with the cytoplasmic membrane and the cell wall of daptomycin resistant enterococci derived from in vitro PK/PD models. Under the first aim, a well established in vitro model of simulated endocardial vegetations (previously validated against an animal endocarditis model) will be used to determine the breakpoints (and corresponding doses) utilizing various clinical doses of daptomycin. The daptomycin resistant strains developed in this model with daptomycin exposure will then be examined for changes in the cell wall, cytoplasmic membrane, gene sequences and gene expression. The proposed research is innovative because we will utilize an in vitro PK/PD model, which both simulates drug pressure under clinical conditions and allows for frequent assessment of changes in the organism, to derive the daptomycin resistant mutants to be studied. The research proposed in this application is significant because it is expected to provide the knowledge needed to understand the resistance characteristics of enterococci and their relationship to daptomycin dose exposure that will lead to dose optimization and improved patient outcomes. Once such knowledge is available, improved patient outcomes and the preservation of daptomycin as a viable therapeutic option for the treatment of enterococcal infections will result.